Identification and characterization of cell surface major vault protein (csMVP) in circulating tumor cells (CTCs) derived from liver cancer patients = 순환종양세포 표면에서 발현되는 major vault protein (MVP) 단백질의 동정 및 기능연구

다국어 초록 (Multilingual Abstract)

Circulating tumor cells (CTCs) in peripheral blood play a major role in the initiation of hepatocellular carcinoma (HCC) metastasis and recurrence even after curative treatments. Major vault protein (MVP) is upregulated during malignant progression and drug resistance in various cancer cells. MVP has been known as a cytonuclear protein, but we found that MVP is expressed on the surface of HCC cells but not on the surface of normal hepatocytes. MVP knockdown decreases HCC cell growth and increases cell death, and treatment with anti-MVP antibodies (α-MVPs) recognizing cell surface MVP (csMVP) inhibits HCC cell proliferation, suggesting that csMVP contributes to HCC cell proliferation. The expression of csMVP is induced under stressful environments, and cell sorting revealed that csMVP (+) HCC cells have a higher clonogenic survival than csMVP (-) HCC cells. Treatment with α-MVP inhibits clonogenic survival and invasive and migratory potential of HCC cells, suggesting that csMVP contributes to cell survival, invasion and migration. csMVP-associated signaling also supports for the role of csMVP in the survival and metastasis of HCC cells. csMVP (+) CTCs are detected in HCC patients (approximately 88%) but not in healthy donors, and the number of csMVP (+) CTCs is further increased in cancer patients with liver metastases. csMVP is only detected in CTCs with mesenchymal phenotype or intermediate phenotype with neither epithelial nor mesenchymal markers. The results suggest that csMVP is a novel surface marker on CTCs with nonepithelial phenotype and promotes cancer progression through improving survival of CTCs in HCCs.

국문 초록 (Abstract)

암 환자의 혈액에 존재하는 순환종양세포(Circulating tumor cells, CTCs)은 간암의 초기 전이나 재발에 중요한 역할을 한다. Major vault protein (MVP)는 다양한 암세포에서 악성으로 진행되고, 약물의 저항성 유발시 발현이 증가한다. MVP는 현재까지 세포질과 핵 단백질로 알려져 있었으나, 본 논문에서는 정상 간세포의 표면에는 발현하지 않으나, 다양한 간암세포 표면에서 발현하는 MVP를 동정 및 특성을 분석하였다. MVP를 결핍시킨 간암세포에서 세포 주기(Cell cycle)이 G2/M기에 정지되어 간암세포의 증식이 크게 감소되고, 세포 사멸도 크게 증가하였다. 세포 표면 MVP 항체를 처리해 본 결과 역시 간암세포의 증식이 억제되었다. 이러한 결과는 csMVP가 간암세포의 증식에 관여한다는 것을 알 수 있었다. csMVP(+) 간암세포와 csMVP(-) 간암세포를 분리하여 clonogenic survival을 분석한 결과 csMVP(+) 간암세포의 생존율이 csMVP(-) 간암세포의 생존율 보다 높게 나타났다. 동일 실험을 MVP항체를 처리한 것과 처리하지 않은 것으로 진행하였을 때 MVP항체를 처리한 군에서 생존율이 크게 떨어지는 것을 확인 하였으며, Migration과 Invasion 역시 크게 감소하는 것을 확인 할 수 있었다. 이러한 결과를 토대로 csMVP가 간암세포의 생존과 Migration 및 Invasion에 크게 관여하는 것을 알 수 있었다. 이러한 결과를 토대로, csMVP는 종양형성과 전이에 있어서 중요한 역할을 하고 있으며 FAK/mTOR 신호를 통해 간암의 성장과 전이를 조절 하는 것이라 생각된다. csMVP는 간암환자 혈액에 존재하는 CTC는 검출하지만, 정상인의 혈액세포에는 발현하지 않는다. 또한 타장기에서 간으로 전이된 암환자의 혈액에서 csMVP(+)-CTCs의 수가 크게 증가함을 알 수 있었다. 추가적으로, 이러한 csMVP(+)-CTCs가 중간엽성 (mesenchymal) 표현형을 보이는 것을 확인 할 수 있었다. 이러한 결과는 csMVP가 중요한 비상피성 표현형을 갖는 CTCs의 마커이며, 또한 csMVP(+)-CTCs는 암의 진행 및 생존에 있어서 중요한 역할을 할 것이라 제안 할 수 있겠다.

목차 (Table of Contents)

• Ι . ABSTRACT 1
• Ⅱ. Introduction 3
• Ⅲ. MATERIALS AND METHODS 6
• 1. Cells, tumor sphere culture, and drug treatment 6
• 2. Patients and sample collection 7
• 3. Antibody biotinylation 7
• 4. Enrichment of CTCs by depletion of CD45-postive cells 7
• 5. Immunocytochemistry 8
• 6. Spiking assay 11
• 7. Cell surface biotinylation, immunoprecipitation, and Western Blotting 11
• 8. Mass spectrometry 12
• 9. Western Blotting of signaling molecules 13
• 10. In vitro cell invasion and migration assays 14
• 11. siRNAs 14
• 12. Flow cytometry and cell sorting 15
• 13. Apoptosis and cell viability assay 15
• 14. Clonogenic survival assay 15
• 15. BrdU incorporation assay 16
• 16. CTCs RNA extraction and next generation sequencing 17
• 17. Statistical analysis 17
• Ⅳ. RESULTS
• 1. MVP is immunoprecipitated with CTC-reactive MAb 63-B6 18
• 2. csMVP is expressed on hESCs and HCC cells but not on normal hepatocytes 29
• 3. MVP contribute to cell proliferation and survival 31
• 4. MVP depletion arrest cell cycle at G2/M phase 37
• 5. MVP-mediated regulation of the FAK, ERK, AKT, mTOR pathway supports proliferation and survival of HCC cells 41
• 6. csMVP expression in HCC cells enhanced under stressful environments 44
• 7. Cell surface expression of MVP contributes to HCC cell proliferation and survival 47
• 8. csMVP contributes to detachment, invasion and migration of HCC cells from the primary tumor 52
• 9. csMVP function is mediated through the control of ERK, Akt, S6K and FAK signaling 56
• 10. ERK/mTOR pathway regulated csMVP expression 59
• 11. Detection of csMVP-positive CTCs is highly sensitive according to the cell spiking assay 62
• 12. Detection of csMVP-positive CTCs from healthy volunteers, hepatitis, HCC and secondary liver cancer patients 64
• 13. csMVP-positive CTCs do not belong to circulating cells with macrophage phenotype 79
• 14. csMVP-positive CTCs show mesenchymal phenotype or intermediate phenotype with neither epithelial nor mesenchymal phenotype 81
• 15. Next generation RNA Sequencing of csMVP positive- or negative-CTCs derived from metastatic cancer patients 86
• Ⅴ. DISCUSSION 92
• Ⅵ. REFERENCE 99